Increased permeability to Ca2+ in response to a depolarizing membrane potential occurs in a wide variety of excitable cell types. In some cell this permeability increase is either blocked or enhanced by dihydropyridines, leading to the hypothesis that the dihydropyridine binding site is part of or associated with the voltage-dependent calcium channel. We propose to purify the DHP binding protein from bovine ventricular tissue and to characterize its subunit composition. This will be accomplished using the binding radioactive DHPS to follow the purification. Purification procedures will utilize conventional column chromatography, HPLC and FPLC techniques, affinity chromatography, and ultracentrifugation. In a collaborative effort, we will use reconstitution techniques to establish whether the DHP binding protein is also the voltage- dependent calcium channel. Important to this purification and characterization of the cardiac DHP binding protein will be the production of antibody and toxin probes specific for this protein. One approach to the preparation of specific antibodies will be to use the subunits of the skeletal muscle DHP binding protein as antigens. We will also use the partially-purified cardiac preparation. Probes of the DHP binding protein will be used to purify it further, to determine the subunit composition, and to analyze the arrangement of the subunits in the membrane.